Exon-4 Mutations in KRAS Affect MEK/ERK and PI3K/AKT Signaling in Human Multiple Myeloma Cell Lines

Abstract

Approximately 20% of multiple myeloma (MM) cases harbor a point mutation in KRAS. However, there is still no final consent on whether KRAS-mutations are associated with disease outcome. Specifically, no data exist on whether KRAS-mutations have an impact on survival of MM patients at diagnosis in the era of novel agents. Direct blockade of KRAS for therapeutic purposes is mostly impossible, but recently a mutation-specific covalent inhibitor targeting KRAS^p.G12C entered into clinical trials. However, other KRAS hotspot-mutations exist in MM patients, including the less common exon-4 mutations. For the current study, the coding regions of KRAS were deep-sequenced in 80 newly diagnosed MM patients, uniformely treated with three cycles of bortezomib plus dexamethasone and cyclophosphamide (VCD)-induction, followed by high-dose chemotherapy and autologous stem cell transplantation. Moreover, the functional impact of KRAS^p.G12A and the exon-4 mutations p.A146T and p.A146V on different survival pathways was investigated. Specifically, KRAS^WT, KRAS^p.G12A, KRAS^p.A146T, and KRAS^p.A146V were overexpressed in HEK293 cells and the KRAS^WT MM cell lines JJN3 and OPM2 using lentiviral transduction and the Sleeping Beauty vector system. Even though KRAS-mutations were not correlated with survival, all KRAS-mutants were found capable of potentially activating MEK/ERK- and sustaining PI3K/AKT-signaling in MM cells.

Keywords: AKT-signaling; KRAS; MEK/ERK-signaling; amplicon sequencing; multiple myeloma.

Figure 1

Distribution of KRAS mutations in the MM cohort studied, and also including two MM cell lines (AMO1, MM1.S) with known KRAS-mutations [26], (A) and within the CoMMpass dataset which was generated as part of the Multiple Myeloma Research Foundation Personalized Medicine Initiatives (Initiatives 2014) [25]; (B). Additionally shown is the biological relevance of the KRAS-mutations detected in the current cohort according to the bioinformatic predictor tools GERP, PolyPhen and PhastCons. (A) The variant allele frequency is given as an approximate indicator of the level of clonality within the sample (A,B). The A146V mutation which was detected in a MM patient of the DSMM cohort was assigned to the group of clonal KRAS-mutations (see Figure S1). n: number, MM: multiple myeloma, AA: amino acid, SNV: single nucleotide variant.

Figure 2

Kaplan Meier plots, showing the difference in overall survival, event free survival, and progression free survival between MM cases with or without KRAS mutation.

Figure 3

pLenti6.3 EmGFP-KRAS^{WT/p.G12A/p.A146T/p.A146V} expression in HEK293 cells as demonstrated by Western blot and fluorescence microscopy analysis. All KRAS-constructs are predominantly localized to the cell membrane of HEK293 cells compared to the pLenti6.2-EmGFP control 24 h after transfection. Fluorescence microscopy analysis was performed at 400× magnification. The corresponding original Western blots are shown in Figure S2. The raw intensities and the intensity ratios for each band are listed in Table S2.

Figure 4

Representative expression and activation-levels of RTK effectors after transfection of KRAS^WT and KRAS^mut constructs into HEK293 cells. The corresponding original Western blots are shown in Figure S3. The raw intensities and the intensity ratios for each band are listed in Table S3.

Figure 5

Representative expression and activation levels of RTK effectors upon the overexpression of KRAS^WT and mutant KRAS in MM cells, following lentiviral transfection of JJN3 and OPM2 cells (A). Stable transposition with Sleeping Beauty vectors of JJN3 and OPM2 cells, (B) or stable transposition with Sleeping Beauty vectors in OPM2 cells, which were either harvested from normal culture medium (Ø PBS) or after a 40 min washout period with PBS to curb FBS-mediated extracellular signals (40 min PBS), (C). Note: The same sample preparation was used for the blots shown for OPM2 in (B,C) (left part). The pictures for AKT and phospho-AKT staining are used in both figures. The corresponding original Western blots are shown in Figure S4A–C. The raw intensities and the intensity ratios for each band are listed in Table S4A–C. pLenti6.2: JJN3 or OPM2 cells transfected with the empty vector pLenti6.2. pT2-CAG-puro: JJN3 or OPM2 cells transfected with the empty vector pT2-CAG-puro.

Figure 6

Experimental approaches to investigate the functional role of the KRAS exon-4 mutations p.A146T and p.A146V in comparison to the exon-2 mutation p.G12A.